The yeast plasma-membrane H+-ATPase is encoded by the PMA1 gene and belongs to an actively studied group of cation pumps known as P2-ATPases. Other members of the group include the Na+, K+-, H+, K+-, and Ca2+-ATPases animal cells, which create the ion gradients that drive essential physiological processes ranging from nutrient uptake and cell volume regulation to muscle contraction and propagation of the nerve action potential. Like these close relatives, the 100 kDa yeast polypeptide is organized into a membrane domain, with ten membrane-spanning helices that assemble together to form the transport channel, and three cytoplasmic domains, where ATP is bound and hydrolyzed. The long-range purpose of this project is to build a detailed picture of H+-ATPase function, biogenesis, and regulation. The next grant period will have the following aims: (1) to dissect the coupling between ATP hydrolysis and H+ transport by focusing on the major E1-E2 conformational change that dominates the reaction cycle. More specifically, a combination of scanning mutagenesis and fluorescent labeling will be used to probe structure-function relationships throughout several of the "stalk" segments that connect the cytoplasmic and membrane parts of the ATPase. Underlying this approach is the recent discovery of a remarkable cluster of mutations in one stalk segment that shift the E1-E2 equilibrium, leading to an easily recognized vanadate-resistant phenotype. (2) A second part of the project will map and analyze a group of six to eight Ser/Thr residues that undergo stepwise phosphorylation as newly synthesized H+-ATPase moves from the ER to the plasma membrane. The goal will be to pinpoint the secretory compartment within which each phosphorylation event occurs and to learn whether phosphorylation is essential for ATPase trafficking and/or activity. (3) Finally, a third group of experiments will explore the molecular mechanism by which glucose activates the H+-ATPase, focusing on interactions between the C-terminus (thought to function as a regulatory domain) and catalytically important parts of the polypeptide. Taken together, these studies will yield information about key structure-function relationships within the H+-ATPase polypeptide, and will also help to understand the mechanism by which newly synthesized ATPase matures and is delivered to the plasma membrane. Given the clear-cut similarities between the yeast H+-ATPase and other P2-ATPases, the results should provide useful insights into the group as a whole. [unreadable] [unreadable]